Search for Genes Potentially Involved inMycobacterium tuberculosisVirulence by mRNA Differential Display

Abstract

An mRNA differential display (DD) assay was developed to compare gene expression betweenMycobacterium tuberculosisH37Rv and its avirulent mutant H37Ra. The DD protocol made use of an oligo(dT) to prime reverse-transcriptase (RT)-dependent transcription of poly-A tailed mRNAs and a PCR amplification of the RT products by using ten 12-base arbitrary primers in all their pair combinations. This analysis yielded 745 and 708 bands, including 52 and 15 differentially generated bands, in the strains H37Rv and H37Ra, respectively. Six cDNAs that appeared to be expressed in H37Rv, but not in H37Ra, were reamplified and cloned and at least 10 inserts were sequenced for each cloned cDNA. After resolving discrepant results, 6 inserts were found highly homologous toM. tuberculosisH37Rv genes. Three of these,i.e.,genes Rv2770c, Rv1345, and Rv0288, coding respectively for a member of the PPE protein family, a probable polyketide synthase, and a member of the protein family containing ESAT-6, have been predictively associated to immunological or pathogenetic aspects ofM. tuberculosisinfection; the other genes,i.e.,Rv2336, Rv1320c, and Rv2819c, code for proteins with unknown functions. These results show that mRNA DD methodology can represent a potential tool for investigation ofM. tuberculosisgene expression.